(a) Field of the Invention
The present invention relates to methods for detecting and treating protein trafficking disorders.
(b) Description of the Prior Art
The quality control system of the endoplasmic reticulum (ER) ensures that only folded proteins proceed further along the secretory pathway. Some of the abundant ER proteins are components of molecular chaperone systems which bind to unfolded proteins, retaining them in the ER. Enzymes which act to fold other proteins are also abundant in the ER and comprise several disulfide isomerases and prolyl peptidyl isomerases (Freedman et al., Trends Biochem Sci. 19:331-336 (1994)). How chaperones and folding enzymes interact to facilitate protein folding in the ER is not known.
Calnexin and calreticulin participate in a molecular chaperone system which integrates the processes of N-glycosylation and quality control (Bergeron et al., Trends BioChem Sci., 19:124-128 (1994) and Helenius et al., Trends Cell Biol., 7:193-200 (1997). They both are lectins that bind N-linked glycans of the form GlcNAc2Man9Glcl which result from the removal of the two outer glucoses from a GlcNAc2Man9Glc3 oligosaccharide by the sequential action of glucosidases I and II. Removal of the innermost glucose by glucosidase II prevents binding by calnexin and calreticulin. Then, if the glycoprotein is unfolded, a glucose residue is added back to the high mannose core by the enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) which recognizes unfolded proteins (Sousa et al., EMBO J., 14:4196-420 (1995)3). Consequently, during folding glycoproteins undergo cycles of binding and release from calnexin and calreticulin which are driven by the addition and removal of a glucose residue (Helenius et al., Trends Cell Biol., 7:193-200 (1997)). As a result of the specificity of UGGT, only unfolded glycoproteins bind to calnexin and calreticulin in vivo (Ou et al., Nature, 364:771-776 (1993) and Peterson et al., Mol. Biol. Cell, 6:1173-1184 (1995) ), even though these lectins do not recognize the conformation of their protein substrates (Rodan et al., EMBO J., 15:6921-6930 (1996) and Zapun et al., Cell, 88:29-38 (1997)).
Monoglucosylated glycoproteins, in addition to binding to calnexin and calreticulin, can also apparently be cross-linked to the ER protein ERp57 (Oliver et al., Science, 275:86-88(1997) and Elliott et al., J. Biol. Chem., 272:13849-13855 (1997)) (also known as ER-60, ERp60, ERp61, GRP58, P58, HIP-70 or Q2; (Elliott et al., J. Biol. Chem., 272:13849-13855 (1997). ERp57 is homologous to protein disulfide isomerase (PDI) and has been shown to exhibit thiol-disulfide oxidoreductase activity in vitro (Bourdi et al., Arch. Biochem. Biophys., 323:397-403 (1995)).
Considering the impact of protein trafficking disorders and the lack of therapies to control protein trafficking disorders, it would be advantageous to be provided with methods to effectively diagnose and treat these disorders.
It would also be advantageous to be provided with methods for folding proteins into their biologically active correctly folded form.
It is an aim of the present invention to provide methods of treating and diagnosing protein trafficking disorders and controlling secretory protein production.
Another aim of the present invention is to provide methods for folding unfolded proteins, especially in heterologous expression systems.
In accordance with the present invention, there is provided methods for folding unfolded proteins or for enhancing the formation of folded proteins which comprise exposing an unfolded protein to a biological preparation comprising ERp57 in combination with calnexin or calreticulin under conditions to permit folding of the unfolded protein.
In accordance with the present invention, there is provided a kit for converting unfolded proteins to folded proteins or for enhancing the formation of folded proteins which comprises:
a) at least one of calnexin or calreticulin;
b) ERp57;
c) UDP-glucose:glycoprotein glucosyltransferase (UGGT);
d) the unfolded protein;
e) UDP-glucose; and
f) an appropriate buffer.
The enzymes, calnexin and/or calreticulin can be fixed to a solid surface. For example, a carrier.
In accordance with the present invention, there is provided methods for forming disulfide bonds or for enhancing the formation of disulfide bonds in an unfolded protein which comprises the step of exposing an unfolded protein to a biological preparation comprising ERp57 in combination with calnexin or calreticulin under conditions to permit folding of the unfolded protein.
In accordance with the present invention, there is provided methods of increasing secretory protein production in a biological preparation, comprising administering an ERp57 suppressor agent to a biological preparation in an amount effective to increase secretory protein production.
In accordance with the present invention, there is provided agents which decrease ERp57 cooperation with calnexin or calreticulin for use in the manufacture of a medicament for increasing secretory protein production in a warm-blooded animal.
In accordance with the present invention, there is provided compositions which decrease ERp57 cooperation with calnexin or calreticulin for use in the manufacture of a medicament for treating a warmblooded animal for protein trafficking disorders which require reduction of ERp57 cooperation with calnexin or calreticulin.
In accordance with the present invention, there is provided compositions that include an agent which stimulates ERp57 cooperation with calnexin or calreticulin for use in the manufacture of a medicament for treating a warm-blooded animal for protein trafficking disorders which require stimulation of ERp57 cooperation with calnexin or calreticulin.
In accordance with the present invention, there is provided conjugates to the endoplasmic reticulum for use in the manufacture of a medicament for treating a warm-blooded animal for protein trafficking disorders.
In accordance with the present invention, there is provided methods of diagnosing a protein trafficking disorder in a warm-blooded animal, comprising the steps of:
a) exposing an anti-ERp57 antibody containing a reporter group to the ER of a warm-blooded animal under conditions and for a sufficient time to permit binding to ERp57;
b) detecting the amount of ERp57; and
c) determining therefrom the presence of a protein trafficking disorder.
In accordance with the present invention, there is provided methods of diagnosing a protein trafficking disorder in a biological preparation, comprising the steps of:
a) exposing an anti-ERp57 antibody containing a reporter group to the biological preparation under conditions and for a sufficient time to permit binding to ERp57;
b) detecting the amount of ERp57; and
c) determining therefrom the presence of a protein trafficking disorder.